Hesty Wahyuningsih1, Ferdy K Cayami1, Udin Bahrudin2, Mochamad A Sobirin3, Farmaditya Ep Mundhofir1, Sultana Mh Faradz1, Ichiro Hisatome4. 1. Center for Biomedical Research, Diponegoro University Faculty of Medicine, Semarang, Indonesia. 2. Center for Biomedical Research, Diponegoro University Faculty of Medicine, Semarang, Indonesia; †Department of Cardiology and Vascular Medicine, Diponegoro University Faculty of Medicine, Semarang, Indonesia. 3. †Department of Cardiology and Vascular Medicine, Diponegoro University Faculty of Medicine, Semarang, Indonesia; ‡Department of Pharmacology and Therapeutic, Diponegoro University Faculty of Medicine, Semarang, Indonesia. 4. §Division of Regenerative Medicine and Therapeutics, Department of Genetic Medicine and Regenerative Therapeutics, Institute of Regenerative Medicine and Biofunction, Tottori University, Yonago 683-8503, Japan.
Abstract
BACKGROUND: High resolution melting (HRM) is a post-PCR technique for variant screening and genotyping based on the different melting points of DNA fragments. The advantages of this technique are that it is fast, simple, and efficient and has a high output, particularly for screening of a large number of samples. APOA1 encodes apolipoprotein A1 (apoA1) which is a major component of high density lipoprotein cholesterol (HDL-C). This study aimed to obtain an optimal quantitative polymerase chain reaction (qPCR)-HRM condition for screening of APOA1 variance. METHODS: Genomic DNA was isolated from a peripheral blood sample using the salting out method. APOA1 was amplified using the RotorGeneQ 5Plex HRM. The PCR product was visualized with the HRM amplification curve and confirmed using gel electrophoresis. The melting profile was confirmed by looking at the melting curve. RESULTS: Five sets of primers covering the translated region of APOA1 exons were designed with expected PCR product size of 100-400 bps. The amplified segments of DNA were amplicons 2, 3, 4A, 4B, and 4C. Amplicons 2, 3 and 4B were optimized at an annealing temperature of 60 °C at 40 PCR cycles. Amplicon 4A was optimized at an annealing temperature of 62 °C at 45 PCR cycles. Amplicon 4C was optimized at an annealing temperature of 63 °C at 50 PCR cycles. CONCLUSION: In addition to the suitable procedures of DNA isolation and quantification, primer design and an estimated PCR product size, the data of this study showed that appropriate annealing temperature and PCR cycles were important factors in optimization of HRM technique for variant screening in APOA1.
BACKGROUND: High resolution melting (HRM) is a post-PCR technique for variant screening and genotyping based on the different melting points of DNA fragments. The advantages of this technique are that it is fast, simple, and efficient and has a high output, particularly for screening of a large number of samples. APOA1 encodes apolipoprotein A1 (apoA1) which is a major component of high density lipoprotein cholesterol (HDL-C). This study aimed to obtain an optimal quantitative polymerase chain reaction (qPCR)-HRM condition for screening of APOA1 variance. METHODS: Genomic DNA was isolated from a peripheral blood sample using the salting out method. APOA1 was amplified using the RotorGeneQ 5Plex HRM. The PCR product was visualized with the HRM amplification curve and confirmed using gel electrophoresis. The melting profile was confirmed by looking at the melting curve. RESULTS: Five sets of primers covering the translated region of APOA1 exons were designed with expected PCR product size of 100-400 bps. The amplified segments of DNA were amplicons 2, 3, 4A, 4B, and 4C. Amplicons 2, 3 and 4B were optimized at an annealing temperature of 60 °C at 40 PCR cycles. Amplicon 4A was optimized at an annealing temperature of 62 °C at 45 PCR cycles. Amplicon 4C was optimized at an annealing temperature of 63 °C at 50 PCR cycles. CONCLUSION: In addition to the suitable procedures of DNA isolation and quantification, primer design and an estimated PCR product size, the data of this study showed that appropriate annealing temperature and PCR cycles were important factors in optimization of HRM technique for variant screening in APOA1.
Authors: Zhiping Wu; Matthew A Wagner; Lemin Zheng; John S Parks; Jacinto M Shy; Jonathan D Smith; Valentin Gogonea; Stanley L Hazen Journal: Nat Struct Mol Biol Date: 2007-08-05 Impact factor: 15.369
Authors: G Kees Hovingh; Eric de Groot; Wim van der Steeg; S Matthijs Boekholdt; Barbara A Hutten; Jan Albert Kuivenhoven; John J P Kastelein Journal: Curr Opin Lipidol Date: 2005-04 Impact factor: 4.776